In October, 2011, Ronald S. Petralia and Ya-Xian Wang moved from the original project of Dr. Robert Wenthold, The Molecular Mechanisms of Synaptic Transmission, to the newly-established Advanced Imaging Core facility of NIDCD. This core is dedicated primarily to collaborating with and training others scientists in NIDCD in studies utilizing electron microscopy either alone or in conjunction with light microscopy. Also, to a limited extent, some work is done with other NIH institutes or outside NIH. Many of the projects reported here were continuations of those described in the FY11 Annual Report of the Molecular Mechanisms of Synaptic Transmission project, and this has been referred to also in the final (FY12) report of that project. The specific research projects (collaboration and/or training) that the Core has been involved in this year are described below. In addition to these projects, the Core generally oversees common equipment (microscopes, freezers, biological microwave oven, lockers, culture room, etc.) and space of the NIDCD PIs in building 50. Also, Ronald Petralia regularly reviews papers for professional journals and reviews many various documents for NIDCD PIs, reviews grants, writes review papers and letters of recommendation, serves on several NIH committees and helps coordinate laboratory and office safety efforts for NIDCD. Projects: In calendar year 2012 so far, the Core has 9 research papers published or in press, as well as 2 reviews. NIDCD: Matt Kelley/Richard Chadwick NIDCD labs: The Core works with fellow, Kate Szarama (Ph.D. Graduate Student), on mechanical properties of developing pillar cells in comparison to outer hair cells, in the mouse cochlea; this project has involved the Core in active research and extensive training. The first paper, on the cytoskeletal changes in actin and microtubules that underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea, is now published in the journal, Development. An additional related paper related is in review. A third paper is in review, and looks at hypothyroid pillar cells and the effect of thyroid hormone secretion on Fgf receptor. We now are looking at the relatively radical changes in the ultrastructure of pillar cells during early postnatal development and the functional implications of these changes. The Core performed extensive immunogold labeling and EM, while Kate was in Sweden, and this work followed up on Kates initial work with the Core on this. Now Kate has started working with the Core regularly on this project. Matt Kelley NIDCD lab: Work with fellow, Helen May-Simera, on the ciliary proteins, BBS8 and IFT20, as part of a study on 'ciliary' proteins and their association with the PCP protein Vangl2; This study began in early 2011. For this project, the Core has been performing EM immunogold labeling plus electron microscopy on both the developing mouse cochlea as well as on the ventricular zone of the rat brain, concentrating on labeling patterns in kinocilia. For this study, the Core also has been involved in the extensive cochlea tissue preparation, in collaboration with Kate Szarama. It is not clear yet how much work will be done on this project in the near future. Bechara Kachar lab: The Core has been involved in 3 projects in progress with Dr. Kachars lab during the designated time period of this report. The first is with Taro Fujikawa on the kainate receptors of the outer hair cells and their afferent terminals; it has involved both training and extensive performance of research. The second project is with Janaina Brusco, on EM ultrastructure of synapses, especially related to GABAergic terminal distribution, in the posterodorsal medial amygdala. The core recently performed additional EM studies for this. The third project is directly with Bechara and involves the examination of specially prepared auditory and vestibular tissue to be used for tomography or other special EM techniques. The Core expects to become more deeply involved in this study and also expand the versatility of the Core by learning new methodologies from Bechara. The Core also gives occasional help with the projects of other staff of Becharas lab. Tom Friedman/Inna Belyantseva lab: The Core has been training Inna and assisting her with tissue preparation and interpretation of data involving electron microscopy. Stephan Brenowitz lab: Last year, the Core performed some studies of immunogold labeling in the dorsal cochlear nucleus in relation to Stephans projects. Recently, this involvement has increased, with the Core now involved in 2 major projects with 2 postdoctoral fellows in the laboratory, Milos Sedlacek and Shan He. Shan recently presented some of this work at a Gordon Research Conference. Vijay Camasamudram/Yong Zeng/Ron Bush NIDCD/NEI lab: The Core has been preparing tissue and studying sections with EM for a study on changes in immunolocalization of a protein, in relation to treatment of a retinal disease. The Core also published a paper in the Journal of Biological Chemistry in early 2012 on a study done with Gail Seabold (Robert Wenthold (NIDCD)/Sharon Milgram (NHLBI) labs), on synaptic adhesion-like molecule 1 (SALM1) trafficking in neurons, We also have worked together on some associated studies. Other NIH institutes: NIA Mattson Lab/Mark Mattson and Pamela Yao lab: The Core has been involved in 2 projects with these scientists: one on the role of sonic hedgehog in synapse development and function in postmitotic neurons (5 papers recently published, in press, or in review, + posters presented at meetings); another looks at the role of endocytic accessory proteins, especially AP180 and Calm, in neurons. The Core recently adapted TSA (tyramide signal augmentation) to enhance EM labeling of terminals in hippocampal neuron cultures transfected with AP180 or Calm constructs. NICHD Chris McBain/Ken Pelkey/Megan Wyeth lab: Recently, the Core has begun work on a project looking at the role of Neto1/2, a kainate receptor auxiliary protein, in hippocampal circuitry. This involves work of the Core on preparation of tissue (KO and WT mice), immunogold labeling, and EM, as well as training of the fellow, Megan. We will have a poster on this at the Society for Neuroscience meeting. NICHD Andres Buonanno lab: We have collaborated in past years and now we are beginning a collaboration involving EM immunolocalization of antibodies to some neuregulin-associated proteins. NINDS Katherine Roche lab: The Core assisted a graduate student, Stephanie Payne, with interpretation of localization patterns in brain structures of a beta-gal KI reporter mouse, helping with tissue preparation, training, and interpretation of localization patterns. NHLBI John Hammer lab: We have been studying the localization of the motor protein, myosin My18A in the cerebellum, utilizing postembedding immunogold and preembedding immunoperoxidase methods. That lab will present a poster on this collaboration at the ASCB meeting. Outside NIH: 1) Johns Hopkins - Paul Worley lab: Last year the Core worked on several projects for this lab, and published 2 papers last year and a third this year (in Cell, Nat Neurosci). Now the Core is collaborating with Paul and Po Chen on a molecule called Rheb1, an immediate early gene that encodes for a small GTPase that activates a kinase that is a mammalian target of rapamycin (mTOR). 2) Gail Seabold: In addition to the published paper (J Biol Chem), Gail has worked with the Core on some subsequent studies on SALMs. We also hope to collaborate with some outside scientists looking at the role of SALMs in human disease. 3) Also, there are some earlier collaborations that were published recently (in Nat Meth, Neuron) or are in preparation or review.